Sterols attract increasing attention due to their important bioactivities. The oleaginous yeastYarrowia lipolyticahas large lipid droplets, which provide storage for the accumulated steroid compounds. In this study, we have successfully constructed a campesterol biosynthetic pathway by modifying the synthetic pathway of ergosterol inY.lipolyticawith different capacity of lipid synthesis.The results showed that the maximal campesterol production was produced in the engineered strain YL-D+M-E-, as the optimal lipid content. Furthermore, we found that campesterol mainly exists in the lipid droplets. The campesterol production was further accumulated through the overexpression of two copies ofdhcr7.Finally, the maximal campesterol production of 837 mg/L was obtained using a 5-L bioreactor in the engineered YL-D+D+M-E-, exhibiting a 3.7-fold increase compared with the initial strain YL-D+E-. Our results demonstrate that the proper promotion of lipid content plays an important role in campesterol biosynthesis inY. lipolytica, and what we found provides an effective strategy for the production of hydrophobic compounds. Key Points center dot Campesterol was biosynthesized by deleting erg5 and introducing heterologous dhcr7. center dot Campesterol production elevated via promotion of lipid content. center dot Campesterol was mainly found in lipid droplets. center dot Promotion of lipid content is an effective strategy to produce hydrophobic compounds.